PAS (Per-ARNT-Sim) protein interaction domains are widely distributed through biology as documented by their presence in a diverse group of over 9000 proteins, including enzymes, transcription factors and ion channels. Within these contexts, PAS domains participate in a combination of intra- and intermolecular interactions necessary for their function. Intriguingly, many of these interactions can be regulated by environmental changes in bound cofactors or artificial ligands that alter the surrounding protein structure. The structural and biochemical mechanisms of PAS function and this regulation are the focus of the proposed research, continuing our prior studies in this area. Given the diversity of settings utilizing these domains, we will determine general features of PAS regulation by comparative biophysical and biochemical studies of representative members of three different classes of PAS-containing proteins. These include: 1). Photoreceptors that use FMN-based PAS domains to control DNA binding activity in response to blue light. Spectroscopic and structural studies will be used to examine how conformational changes in a sensory PAS domain regulate activity. 2). Bacterial histidine ("sensor") kinases, where PAS domains sense a variety of environmental conditions. Biophysical and biochemical studies of PAS domains and full length proteins will be used to address how activating and inactivating mutations regulate kinase activity. The functional effects of these changes will be probed with in vitro enzymatic and in vivo signaling assays. 3). Eukaryotic transcription factors, including human bHLH/PAS proteins. Extending our work on several of these proteins, we will use spectroscopic tools to investigate a domain that can be artificially triggered to switch conformation by surface mutations. We will also examine how these domains can simultaneously bind multiple protein targets using biochemical and structural studies of transcriptional coactivator recruitment. This research will provide fundamental insights into the signaling mechanisms used by PAS domains, particularly in how they work in conjunction with enzymatic and non-enzymatic effectors. This will improve our understanding of PAS-containing proteins and suggest engineering principles for these proteins. Relevance to public health: PAS domains play a central role in human disease, as point mutations in these elements have been correlated with several cancers and cardiac irregularities. These domains are also essential to several bacterial pathways, providing links to microbial pathogenesis. Improved understanding of PAS structure/function links may provide novel routes for diagnosis and intervention in disease processes.